Research ArticleUsnea Acid as Multidrug Resistance (MDR) ReversingAgent against Human Chronic Myelogenous LeukemiaK562/ADR Cells via an ROS Dependent Apoptosis

Wenjing Wang,1,2,3 Shubin Niu,4 Luxin Qiao,1,2,3 Feili Wei,1,2,3 Jiming Yin,1,2,3

ShanshanWang,1,2,3 Yabo Ouyang,1,2,3 and Dexi Chen 1,2,3

1Capital Medical University Affiliated Beijing You An Hospital, Beijing, 100069, China2Beijing Institute of Hepatology, Beijing, 100069, China3Beijing PrecisionMedicine andTransformation Engineering Technology ResearchCenter ofHepatitis and Liver Cancer, 100069, China4School of biomedicine, Beijing City University, No. 6 Huanghoudian Road Haidian District, Beijing, 100094, China

Correspondence should be addressed to Dexi Chen; dexichen@ccmu.edu.cn

Received 10 September 2018; Revised 8 December 2018; Accepted 23 December 2018; Published 11 February 2019

Academic Editor: Adair Santos

Copyright © 2019 WenjingWang et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Purpose. Multidrug resistance (MDR) is a major obstacle in chemotherapy of leukemia treatments. In this paper, we investigatedUsnea Acid (UA) as MDR reversal agent on hematologic K562/ADR cells via ROS dependent apoptosis.Methods. CCK8 assay wasused to measure cell viability rate of K562/ADR. Intracellular reactive oxygen species (ROS) generation, cell cycle distribution,cell apoptosis were measured with flow cytometry, respectively. Proteins related to apoptosis were measured by Western blot.Intracellular Adriamycin accumulation was observed by confocal microscopy and measured by flow cytometry. Results. In vitrostudy showed intracellular Adriamycin accumulation was remarkably increased by UA. Cell viability treated with Adr (4 𝜇M) wasdecreased from 89.8% ± 4.7 to 32% ± 8.9 by combined with UA (4 𝜇M). Adr-induced apoptosis and G1/G0 phase cell cycle arrestwere remarkably increased by UA, as well as, intracellular ROS level. However, MDR reversing activity of UA was inhibited by N-acetyl cysteine (NAC), a ROS scavenger. Conclusion. These data provide compelling evidence that UA is a promising agent againstMDR in leukemia cell line and suggest a promising therapeutic approach for leukemia.

1. Introduction

Leukemia originates from abnormal hematopoietic stem cellswhich can result in a high number of deaths annually [1].Over the past decades, major advances have been achieved inclinical treatment of leukemia. Despite overall improvementin the outcome of conventional leukemia chemotherapy,multiple drug resistance (MDR) is still the major problem inleukemia chemotherapy [2, 3].

Since first time being reported in 1970, MDR has beenextensively studied by a multitude of academic researchers[4–6]. MDR is extremely complicated can be induced by dif-ferent mechanisms. Overexpressing of ABC-transportersis recognized as the main cause of MDR, which is almostpositive in allmalignant tumor cells [7–9].High level of ABC-transporters in leukemia cells may lead to increasing of drug

efflux, decreasing intracellular drug concentration, therebypreventing the antiproliferation activity of chemotherapydrugs [10, 11]. Adriamycin (Adr) belongs to the anthracyclineantibiotic family, displaying strong cytotoxicity and thereforegenerally being used as chemotherapeutic agents in clinicalincluding leukemia [12]. However, expression of MDR 1mRNAor/and overexpression of proteins of ABS-transporterfamily induced MDR challenging Adr treatment againstleukemia [13]. Based on this situation, developing of noveltherapeutic strategies to reverse MDR is extremely importantin the clinical of leukemia therapy.

Usnea Acid (UA), a bioactive lichen secondary metabo-lite, has been investigated as a promising anticancer agentin different cancer cell lines, including hepatocellular carci-noma, breast cancer, nonsmall cell lung cancer, and coloncancer [14]. In vitro study using UA against malignant cells

HindawiBioMed Research InternationalVolume 2019, Article ID 8727935, 7 pageshttps://doi.org/10.1155/2019/8727935

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2 BioMed Research International

suggesting it can induce cell cycle arrest, autophagy, andapoptosis, thereby, has potential to be developed as achemotherapeutic agent [15].

Reactive oxygen species (ROS) are a group of oxygen-containing, short-lived molecules that are highly reactive[16, 17]. Previous research has indicated that overproductionROS can induce apoptosis via opening the mitochondrialpermeability transition pore and thus releasing proapoptoticfactors in leukemia cells [18, 19].

In this paper, we demonstrated that UA may increase theaccumulation ofAdriamycin in hematologicK562/ADR cells,reverse MDR via ROS dependent apoptosis induction.

2. Materials and Methods

2.1. Chemicals. Usnea Acid (UA), Adriamycin, and NACwere all purchased from sigma ((Sigma, St. Louis, MO, USA).

2.2. Cell Culture. Human cell lines (K562/ADR) were ob-tained from ATCC (Manassas, Virginia, USA) and culturedin Gibco� RPMI-1640 complete medium (Thermo FisherScientific, HK, China) containing 10% heat inactivated FBS,100 U/ml penicillin, and 100 mg/ml streptomycin. Beforethe study, K562/Adr cells were cultured in complete culturesolution without Adriamycin for 48hr.

2.3. Adriamycin Accumulation. Adriamycin accumulationwas measured by intensity of fluorescence of Adr. Cells wereseeded into confocal dishes at a density of 5 × 105 and thentreated with UA (4 𝜇M), Adr (4 𝜇M), and UA plus Adr (4𝜇M, respectively) for 48h; medium with same concentrationof DMSO was used as control. After 3 washes with ice-cold PBS, cells were observed under a confocal microscopy(PerkinElmer UltraVIEW VOX, PE, Billerica, MA) anddetected on BD FACSCalibur Cytometry.

2.4. CCK8 Cell Viability Assay. K562/Adr cells were culturedovernight after plated in triplicate wells in 96-well plates (4× 103 cells/well) followed by exposure to different concentra-tions of UA (0, 2, 4, 6, 8, 16, 32, and 64𝜇M), Adr (0, 2, 4, 6, 8,16, 32, and 64𝜇M), and UA plus Adr (0, 2, 4, 6, 8, 16, 32, and64𝜇M, respectively) for 48h. A total of 10 𝜇l CCK-8 reagentswere added to the wells and kept in the incubator for 2-4 hrat 37∘C after incubating. Finally, the absorbance was deter-mined at 450 nm by a SpectraMax M5 Microplate Reader(Molecular Devices Instruments Inc., Sunnyvale, California,USA).

2.5. Cell Cycle Analysis. Cells were pretreated with UA(4𝜇M), Adr (4𝜇M), and UAplus Adr (4𝜇M, respectively) andincubated for 48h before being collected. Medium with sameconcentration of DMSO was used as control group. Afterincubation, cells were washed in PBS, and fixed in ice-cold70% ethanol before being recentrifuged and incubated withRNase A (200 𝜇g/mL) and propidium iodide (PI, 5 𝜇g/mL).Cell cycle distribution was detected on BD FACSCaliburCytometry. Data was analyzed with Cellquest software (BDBiosciences, Franklin Lakes, New Jersey, USA).

2.6. ROS Generation Measurement. Reactive Oxygen SpeciesAssay Kit (KeyGEN BioTECH, Nanjing, China) was used todetect intracellular ROS levels. Exponentially growing cellswere treated with UA (4𝜇M), Adr (4𝜇M), and UA plus Adr(4𝜇M, respectively) and incubated for 48h before harvestingand performed according to the manufacturer’s instructions.The fluorescence of the cells was monitored using flowcytometry (FACSCalibur, BD, Franklin Lakes, New Jersey,USA). ROS production was calculated as the intensity in thefluorescence compared with the control group.

2.7. Flow Cytometric Analysis of Apoptosis. K562/ADR cellswere seeded into 6-well plates at 5 × 105 cells/well. After12h incubation, cells were then treated with UA (4𝜇M), Adr(4𝜇M), and UA plus Adr (4𝜇M, respectively), followed byharvesting at 48h after treatment before being double-stainedwith Annexin V-FITC/PI (KeyGEN BioTECH, Nanjing,China) and subjected to flow cytometry analysis for detectionof apoptosis. 10,000 cells per sample were analyzed by a BDFACSCalibur Cytometry (BD Biosciences, Franklin Lakes,New Jersey, USA) to quantify apoptotic cells (Annexin V-FITC positive cells).

2.8. Western Blot Analysis. K562/Adr cells were seeded into6-well plates at 5 × 105 cells/well. After overnight incubation,they were pretreated with 4 𝜇M Adr plus 4 𝜇M UA for48h before being harvested and washed with ice-cold PBSand then lysed with ice-cold RIPA lysis buffer (KeyGENBioTECH, Nanjing, China) with 1 mmol/L PMSF. Proteinconcentrations were calculated by BCA assay kits (ThermoFisher SCIENTIFIC, Beijing, China). The western blot wasperformed as previously described [20]. Briefly, 20𝜇g of totalprotein was subjected to 12% SDS-PAGE gel and transferredto PVDFmembranes (Millipore, Atlanta, Georgia, US). Afterblocking with 5% defatted milk for 1h, membranes were thenincubated with primary antibody overnight at 4∘C, followedby HRP-labeled secondary antibody at room temperature for1h. Following each step, the membranes were washed fivetimes with PBS-T for 5min. Immunoreactive proteins weredetected using a chemiluminescence reagent by following theuser manual; the GAPDHwas selected as the loading control.

2.9. Statistical Analysis. All data are presented as mean ±standard deviation (SD) of three separate experiments. Datawere evaluated using SPSS for Student’s t-test and subjectedto one-way or two way analysis of variance.

3. Results

3.1. Usnea Acid Effects on the Proliferation and IntracellularAccumulation of K562/ADR Cells. By combining confocalmicroscopy and flow cytometry analyses, we found thatfluorescence intensity of Adr in K562/ADR cells becamemarkedly higher in UA plus Adr group compared withAdr alone (1.75 folder, p < 0.01), indicating intracellularaccumulation of Adr was increased by UA.

The relative cell viability of treated cells was determinedby CCK8 assay. As the results showed in Figure 1(c), cell

BioMed Research International 3

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Figure 1: Effects of UA on cell viability and intracellular accumulation of Adr in K562/ADR cells. Cells were treated with UA(4 𝜇M), Adr UA(4𝜇M), and UA plus Adr (4 𝜇M, respectively) for 48 hr, medium with same concentration of DMSO was used as control. Adr accumulationwas increased by UR observed by confocal microscopy (b) and flow cytometry(a, d). Cell viability was dramatically decrease by UA plus Adrcompare with Adr alone(c). Columns, values are expressed as mean ± SD.

viability was decreased by combination of UA and AdrcomparedwithUAorAdr alone in a dose-dependentmanner.According to the results of CCK8 assay, cell viability treatedwith Adr (4 𝜇M) was decreased from 89.8% ± 4.7 to 32% ±8.9 by combined with UA (4 𝜇M). Base on that, we selected 4𝜇M as the final concentration to do all the tests.

3.2. Usnea Acid Effects on the Generation of Intracellular ROS,Apoptosis Induction, and Cell Cycle Arresting of K562/ADRCells. With the aim of determining the effective of apop-tosis induction of UA combined with Adr, apoptotic cells

percentage was evaluated by flow cytometric analysis usingAnnexin V/FITC-PI double staining assay. As shown inFigures 2(a) and 2(d), K56/ADR cells were resistant to Adr-induced apoptosis (9.7%); with combination of UA, theapoptotic cells were increased into 20.7%.

To explore the effect of UA combined with Adr on cellcycle distribution, propidium iodide DNA staining flowcytometric analyses were performed. As shown in Figures2(b) and 2(e), UA plus Adr can induce cell cycle arrestedin G1/G0 phase (71.5 %) compare with Adr treatment group(46.3%).

4 BioMed Research International

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Figure 2: UA Plus Adr Induced ROS Generation, Apoptosis, and Cell Cycle Arrest in K562/Adr Cells. Cells were treated with UA(4 𝜇M), AdrUA(4 𝜇M), and UA plus Adr (4 𝜇M) for 48 hr before examination; mediumwith same concentration of DMSOwas used as control. AnnexinV-positive cells were analyzed by flow cytometry (a, d). Cell cycle arresting was observed by PI-staining DNA flow cytometric analysis (b, e).ROS generation was detected by confocal microscopy and flow cytometry (c, f). Columns: values are expressed as mean ± SD.

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Figure 3:MDR Reversing Activity of UAWas Inhibited by NAC. Cells were treated with UA(4 𝜇M), Adr UA(4 𝜇M), and UA plus Adr (4 𝜇M,respectively) for 48 hr before examination; medium with same concentration of DMSO was used as control. Cell viability was detected byCCK8 assay (a). Apoptosis related proteins were analyzed by western blot (b). Intracellular generation of ROS (c) and accumulation of Adr(d) were observed by confocal microscopy and flow cytometry. Columns: values are expressed as mean ± SD.

A great deal of research on multiple different cell lineshas demonstrated that ROS can induce apoptosis [18, 19].It was hypothesized that the primary mechanism of UA-mediated K56/ADR cells sensitization should be the induc-tion of ROS dependent apoptosis. DCFH-DA assay wasused to observe the content of ROS generation. As seenin Figures 2(c) and 2(d), the fluorescence intensity is 1.78-folder higher in UA combination group compared with Adralone.

3.3. Increased Intracellular ROS Level Is Essential for UsneaAcid Reversing Adr Resistance in K562/ADRCells. In order tocharacterize the ROS generation is essential for UA reversingMDR in K562/ADR cells, NAC, a ROS scavenger was intro-duced. We used 10𝜇M DCFH-DA as a fluorescent probe toreact with ROS and measured the intensity of the emittedlight by confocal microscopy and flow cytometry. Accordingto the results, ROS generation enhancement activity of UAwas inhibited by NAC (Figure 3(c)); at the same time, theaugmentation activity of UA on intracellular accumulation ofAdr was reduced (Figure 3(d)).

By using CCK8 assay, we observed that cell viability wassignificantly increased when adding NAC in UA plus Adrgroup (Figure 3(a)), which indicated that enhanced cytotox-icity of Adr by UA was reversed by NAC. Furthermore, wedetected protein expression of cleaved caspase 3 and PARPin K562/ADR cells. As shown in Figure 3(b), cleaved caspase3 and PARP in cotreatment of UA and Adr group weredecreased by NAC.

These results indicated that incubationwithUAenhancedAdr induced apoptosis by regulation of intracellular ROSdependent apoptosis pathway.

4. Discussion

Currently, multidrug resistance (MDR) to antineoplasticdrugs is a tough problem to successful treatment of leukemia.Although the mechanism of MDR has been extensivelystudied by amultitude ofmedical investigators, few drugs thatcan be used in clinical for reversing MDR were developed.Looking for novel agents with anti-MDR activity is thereforeexpected.

Usnea Acid (UA) is a multifunctional bioactive lichensecondary metabolite with potential anti-cancer properties.In vitro anticancer effects of Usnea Acid were shown for thefirst time by Kupchan and Kopperman against Lewis lungcarcinoma [21]. Since then, many other researchers reportedantiproliferative and mitochondrial depressive effects of UAagainst malignant cells in vitro, suggesting its potential use asa chemotherapeutic agent [22–24]. Although the promisingtherapeutic effects of UA have been investigated in differentcancer cell lines, the multidrug resistance reversing activityin leukemia cells has yet to be elucidated. In this study, weinvestigated theMDR reversing activity of UA against humanleukemia Adriamycin- (ADR-) selected multidrug resistance(MDR) cell line K562/ADR.

Most commonly encountered mechanism of multidrugresistance is characterized as intracellular drug depletion by

6 BioMed Research International

effluxpump, leading to a cellular responsiveness. In our study,flow cytometry and confocal microscopy assay showed thatintracellular accumulation of Adr was significantly increasedby UA (Figures 1(a), 1(b), and 1(d)). Results from CCK8 assayindicated that UA can increase Adr antiproliferation activityagainst K562/ADR cells (Figure 1(c)).

Altered cell-cycle checkpoints and apoptosis resistancewere also described asmechanisms ofMDR [25, 26]. By usingflow cytometry, we measured cell-cycle arresting and apop-tosis inducing activity of Adr combined with UA comparedwith Adr alone. As results showed in Figures 2(a), 2(b), 2(d),and 2(e), cocultured with UA, cell-cycle arrested in G1/G0phase by Adr was increase from 46.37% to 71.35%; at thesame time, apoptotic cells induced by Adr increased from9.7% to 20.3%. By combining confocal microscopy and flowcytometry, we found that ROS generation in K562/ADR cellswas significantly increased by UA and Adr coculture (Figures2(c) and 2(f)).

Reactive oxygen species (ROS) is a key stimulator in celldeath. To obtain further information, we use NAC to inhibitROS generation in K562/ADR cells. As results showed inFigure 3, ROS generation and Adr accumulation in K562/ADR cells increased by UA were inhibited by NAC analyzedby confocal microscopy and flow cytometry.

We also found that protein levels of cleaved caspase-3 andcleaved PARP expression in UA and Ader cocultured groupwere decreased by NAC; cell viability was increased at thesame time (Figures 3(a) and 3(b)). These results indicatedthat incubation with UA enhanced ADR induced apoptosisby regulation of ROS generation in K562/ADR cells.

In conclusion, our data indicated that UA possessed thepotential anti-MDR activity in K562/Adr cells through ROS-dependent apoptosis and G1/G0 phase cell-cycle arresting.UA might be a potential therapeutic compound for MDRleukemia treatment. However, there is a need for furtherstudies investigating the molecular signaling mechanismsinduced by UA treatment.

Data Availability

The data used to support the findings of this study areincluded within the article.

Conflicts of Interest

The authors have declared that no conflicts of interest exist.

Acknowledgments

This study was supported by Beijing Natural Science Founda-tion (7174284).

Supplementary Materials

Figure 1. Chemical structure of Usnea Acid and Adriamycin.Figure 2. Docking study of UA into ABCG 2 protein activedomains. (A) Docking position of the binding site of ABCG2, UA is shown as ball and stick mode in blue color. (B) The

two-dimensional ligand-receptor interaction diagram of UAand human homology ABCG 2. (Supplementary Materials)

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